Serum APOC1 levels are decreased in young autoantibody positive children who rapidly progress to type 1 diabetes

Better understanding of the early events in the development of type 1 diabetes is needed to improve prediction and monitoring of the disease progression during the substantially heterogeneous presymptomatic period of the beta cell damaging process. To address this concern, we used mass spectrometry-based proteomics to analyse longitudinal pre-onset plasma sample series from children positive for multiple islet autoantibodies who had rapidly progressed to type 1 diabetes before 4 years of age (n = 10) and compared these with similar measurements from matched children who were either positive for a single autoantibody (n = 10) or autoantibody negative (n = 10). Following statistical analysis of the longitudinal data, targeted serum proteomics was used to verify 11 proteins putatively associated with the disease development in a similar yet independent and larger cohort of children who progressed to the disease within 5 years of age (n = 31) and matched autoantibody negative children (n = 31). These data reiterated extensive age-related trends for protein levels in young children. Further, these analyses demonstrated that the serum levels of two peptides unique for apolipoprotein C1 (APOC1) were decreased after the appearance of the first islet autoantibody and remained relatively less abundant in children who progressed to type 1 diabetes, in comparison to autoantibody negative children.


APOC1 verification results
A discrepancy was noted between the APOC1 LonGP results in the discovery and validation datasets. This can be seen in the representations shown in Supplementary Fig. S2b and Fig.  3a-b. These indicate a "t1d" associated ( Supplementary Fig. S2b) and a "sero" associated effect, (Fig. 3a-b), and reflect differences in the sampling frequency and different aspects of the data. Further analysis and evaluation of this is presented as follows.
For each individual in the discovery and validation datasets, the protein/peptide baseline change after seroconversion was calculated. In the LonGP regression of the discovery dataset, a peak [-7, -5] and trough region [-4, -2], (i.e. 5-7 or 2-4 months prior to diagnosis, Supplementary Fig. S2b) was seen in the progressors group. From the LonGP regression of the validation dataset ( Fig. 3a-b), a general baseline decrease after seroconversion was seen in the progressors group. As further illustrated in Supplementary Fig. S5, the baseline levels of APOC1 decreased more in the progressors group than those of the AAb-group. This change was similarly observed in the discovery dataset ( Supplementary Fig. S6), although was not significant (t-test, p=0.87). Supplementary Fig. S7 further illustrates the baseline difference between the peak and trough region in the validation dataset, which is consistent with a greater difference between the peak and trough regions in the progressor group (t-test, p=0.58 and 0.63).
Due to the superior temporal coverage provided by the validation data, we concluded that the validation data verify that the APOC1 levels in progressors group decrease after seroconversion. Supplementary Table S1. Summary and plasma sample collection of children selected for the discovery proteomics analysis.     Fig. S1. Longitudinal plasma sample collection times of the discovery proteomics study participants. Triangle indicates the date of the appearance of the first autoantibody. Star indicates the date of type 1 diabetes diagnosis. Progressor = child who has developed multiple autoantibodies and progressed to type 1 diabetes before 4 years of age. 1AAb+ child = single autoantibody positive child without progression to type 1 diabetes. AAbchild = autoantibody negative child Progressor 1AAb+ child

SUPPLEMENTARY TABLES
AAb-child Supplementary Fig. S2. Discovery proteomics results of the disease-associated proteins selected for the verification study using targeted proteomics. (a) We first selected proteins that showed differences between the three study groups (with "group" and "group*age" terms in LonGP). (b) Then we selected the proteins that exhibited changes associated with the disease diagnosis ("t1d" term in LonGP). (c) Lastly, the selection was made among proteins that showed changes associated with the seroconversion ("sero" term in LonGP). The shaded areas are 95% confidence intervals. Progressors group = children who have developed multiple autoantibodies and progressed to type 1 diabetes before 4 years of age, 1AAb+ group = single autoantibody positive children without progression to type 1 diabetes. AAb-group = autoantibody negative children. a Supplementary Fig. S5. Baseline change after seroconversion (baseline_after -baseline_before) of two APOC1 peptides: (a) EFGNTLEDK and (b) EWFSETFQK in the validation data. For each individual, we calculated the baseline peptide levels before and after the seroconversion by computing the average of data points before and after seroconversion. The seroconversion age of an AAb-is taken from its matched progressor. Progressors (red) = children who have progressed to type 1 diabetes before 5 years of age. AAb-(blue) = autoantibody negative children. Two sample t-test is perform to check the difference between progressors and AAb-children.
b Supplementary Fig. S6: Baseline change after seroconversion (baseline_after -baseline_before ) for APOC1 in the discovery data. For each individual, we calculated the baseline protein levels before and after the seroconversion by computing the average of data points before and after seroconversion. The seroconversion age of an AAb-is taken from its matched progressor. Progressors (red) = children who have developed multiple autoantibodies and progressed to type 1 diabetes before 4 years of age. 1AAb+ (purple) = single autoantibody positive children without progression to type 1 diabetes. AAb-(blue) = autoantibody negative children. Two sample t-test is perform to check the difference between progressors and AAb-children. a b Supplementary Fig. S7. t1d related baseline change (peak -trough) of two APOC1 peptides: (a) EFGNTLEDK and (b) EWFSETFQK in the validation data. Peak region is defined as [-7, -5], i.e. 5 to 7 months before type 1 diabetes diagnosis. Trough region is defined as [-4, -2], i.e. 2 to 4 months before type 1 diabetes diagnosis. For each individual, we calculated the baseline peptide levels for the peak or trough regions by computing the average of data points that fall into the corresponding regions, where only individuals with data points in both regions are included. Progressors (red) = children who have progressed to type 1 diabetes before 5 years of age. AAb-(blue) = autoantibody negative children. (a) iTRAQ ratios of APOC1 scaled to a common reference from eight matched pairs. In addition, there were five pairs with missing APOC1 values who were excluded from the analysis. (b) Scaled label-free intensities from the six matched case-control pairs. Progressors = progressed to type 1 diabetes between 2.2 and 12.1 years of age. AAb-group = autoantibody negative children.